Fortimicins AM and AP derivatives

ABSTRACT

4,N, 2&#39;-N and 4,2&#39;-Di-N-derivatives of fortimicin AM and fortimicin AP are provided by this invention. The compounds are useful as anti-bacterial agents.

BACKGROUND OF THE INVENTION

The aminoglycoside antibiotics are a valuable therapeutic class ofantibiotics which include the kanamycins, gentamicins, streptomycins,sagamicins and the more recently discovered fortimicins. While thenaturally produced patent antibiotics are generally, in themselves,valuable antibiotics, chemical modifications have been found to improvethe activity, either intrinsic activity or activity against resistantstrains or against one or more strains the parent antibiotic is noteffective against. Thus, chemical modification has provided bothalternative therapeutic agents as well as those which are held inreserve because of the resistance problem. And, because of thedevelopment of aminoglycoside-resistant strains and inactivation of theparent antibiotics by R-mediated factors which can develop, the searchfor new therapeutic entities continues.

Further, some of the naturally produced, parent antibiotics, such asfortimicin B and fortimicin E, are primarily useful as intermediates inpreparing derivatives which have more potent antibacterial propertiesthan their weakly active parent antibiotics. The present inventionprovides derivatives of 2 such fortimicins, fortimicins AM and AP.

The fortimicins of this invention are co-produced in the fermentation ofMicromonospora olivoasterospora ATCC No. 21819,31009 or 31010 accordingto the method of Nara et al. U.S. Pat. Nos. 3,931,400 and 3,976,768which disclose the production of fortimicin A and fortimicin B.

Fortimicin AM and fortimicin AP are minor factors which are co-producedwith fortimicin A, fortimicin B and a number of other minor assignedpatent application Ser. Nos. 025,241; 025,243; 025,247; 025,250;025,251; and 025,252 filed of even date herewith and with the minorfactors disclosed and claimed in commonly assigned, copending UnitedStates patent application Ser. Nos. 863,015 and 863,016, both filed Dec.21, 1977.

The 4-N-derivatives and fortimicin B are disclosed in U.S. Pat. No.4,091,032. The 2'-N and 4,2'-di-N-derivatives of fortimicin B andfortimicin E are disclosed in commonly assigned, co-pending U.S. Pat.application Ser. Nos. 863,012 and 863,010, both filed Dec. 21, 1977.

SUMMARY OF THE INVENTION

The present invention provides 4-N, 2'-N and 4,2'-di-N fortimicins AMand AP. The compounds of this invention are useful as antibioticsagainst susceptible gram positive and gram negative bacilli such asEscherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa,Bacillis subtilis, Proteus vulgaris, Shigella sonnei, Salmonella typhiand Klebsiella pneumonia.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The compounds of this invention are prepared from fortimicins Am and Apwhich are represented by Formula I: ##STR1## wherein R₁ is hydrogen orhydroxy and R₂ is hydrogen when R₁ is hydroxy and hydroxy when R₁ ishydrogen.

When R₁ is hydrogen and R₂ hydroxy, the compound is fortimicin AM. WhenR₁ is hydroxy and R₂ is hydrogen, the compound is fortimicin AP.

Fortimicins AM and AP prepared by the fermentation of Micromonosporaolivoasterospora as detailed in Examples 1-4.

The compounds of this invention are 4-N-, 2'-N and 4,2'-di-N-fortimicinderivatives and are represented by Formula II: ##STR2## wherein: R₁ andR₂ are as defined in Formula I, and R₃ and R₄ are the same or differentmembers of the group consisting of hydrogen, acyl, aminoacyl,diaminoacyl, N-loweralkylaminoacyl, N,N-diloweralkylaminoacyl,hydroxy-substituted aminoacyl, loweralkyl, aminoloweralkyl,diaminoloweralkyl, hydroxyloweralkyl, N-loweralkylaminoloweralkyl,aminohydroxyloweralkyl, N,N-diloweralkylaminoloweralkyl,N-loweralkylaminohydroxyloweralkyl,N,N-diloweralkylaminohydroxyloweralkyl and the pharmaceuticallyacceptable salts thereof with the limitation that R₃ and R₄ each cannotbe hydrogen.

The term acyl, as used in the above definitions refers to acyl radicalsof loweralkylcarboxylic acids represented by the formula ##STR3##wherein R is loweralkyl, i.e., acetyl, propionyl, butyryl, valeryl, etc.

The terms aminoacyl, hydroxy-substituted aminoacyl, etc., enumerated inthe definitions of R₃ and R₄ for formula II include, but are not limitedto as will be obvious to those skilled in the art, naturally occuringamino acids such as glycyl, valyl, alanyl, sarcosyl, leucyl, isoleucyl,prolyl, seryl, and like amino acid residues as well as groups such as2-hydroxy-4-aminobutyryl and like groups. The amino acid residuesincluded in the above terms, with the exception of glycyl, can be eitherin the L- or D-configurations or mixtures thereof.

The term "loweralkyl", as used herein, refers to straight or branchedchain alkyl radicals containing from 1 to 6 carbon atoms and includes,but is not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl,iso-butyl, tert-butyl, n-pentyl, 1-methylbutyl, 2,2-dimethylpropyl,n-hexyl, 2-methylpentyl and the like radicals.

The term "pharmaceutically acceptable salts" refers to the non-toxicacid addition salts of the compounds of Formulae I and II which can beprepared either in situ during the final isolation and purification orby separately reacting the free base with a suitable organic orinorganic acid by methods well known in the art. Representative saltsinclude the mono-, di-, tri-tetra, or other per-salts such as thehydrochloride, hydrobromide, sulfate, bisulfate, acetate, oxalate,valerate, oleate, palmitate, stearate, laurate, borate, benzoate,lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate,tartrate, napsylate and like salts.

The antibiotics of Formula II are effective antibacterial agents againstsusceptible or sensitive strains of gram-negative and gram-positivebacilli such as Staphylococcus aureus, Escherichia coli, Pseudomonasaeruginosa, Bacillus subtilis, Proteus vulgaris, Shigella sonnei,Salmonella typhi and Klebsiella pneumoniae. The compounds of Formula IIare administered parenterally, i.e., intravenously, intramuscularly,intraperitoneally or subcutaneously for systemic effect in daily dosagesof from 20 to 40 mg/kg of body weight daily, preferrably from 25 to 30mg/kg of body weight daily based on lean body weight as is good medicalpractice with the aminoglycoside antibiotics and are preferablyadministered in divided dosages. The compounds can also be administeredorally at the above dosages to sterilize the intestinal tract and canfurther be administered in suppository form.

The term "sensitive or susceptible strains" refers to strains of bacillior organisms which have been demonstrated to be sensitive to aparticular antibiotic in a standard in vitro sensitivity test and thusin vitro activity has been established for a particular antibioticagainst a specific strain of a specific organism.

Fortimicins AM and AP are prepared by the fermentation of Micromonosporaolivoasterospora ATCC No. 21819,31009 or 31010 according to the methodsdescribed by Nara et al. in U.S. Pat. Nos. 3,931,400 and 3,976,768 forthe fermentation of fortimicin A and fortimicin B, and set forth inExamples 1-4 for the fermentation and isolation of

The 4-N-acyl fortimicin AM and AP derivatives are prepared following thegeneral procedure used for the preparation of 4-N-acyl derivatives offortimicins having the fortimicin E stereochemistry for the 4-N-positionas disclosed in commonly assigned, co-pending U.S. application Ser. No.863,010, filed Dec. 21, 1977.

Generally speaking, the 4-N-acyl derivatives can be prepared by reacting3 moles of salicylaldehyde with fortimicin AM or AP which results in theformation of 1,2',6'-tri-N-salicylaldehyde Schiff base fortimicin AM orAP. The latter can then be aminoacylated by coupling the Schiff baseintermediate with a variety of activated carboxylic acid derivativessuch as a carboxylic acid anhydride, a carboxylic acid chloride, anactive carboxylic acid ester or a carboxylic acid azide.

The active esters may be conveniently prepared by reacting theappropriate carboxylic acid, RCOOH with, for example1-hydroxybenzotriazole, N-hydroxysuccinimide orN-hydroxy-5-norbornene-2,3-dicarboximide according to the method of M.Fujino et al., Chem Pharm Bull, Japan 22: 1857 (1974) wherein R is asdefined in formula II for acyl and acyl-containing groups.

For example, the Schiff base fortimicin AM can be aminoacylated with anactive ester represented by the formula A-P-Z, i.e.,N-benzyloxycarbonylglycyl-N-hydroxysuccinimide active ester (A═ONS,R═COCH₂ NH--),N-benzyloxycarbonyl-β-alanyl-N-hydroxy-5-norbornene-2,3-dicarboximideactive ester (A═ONB, R═COCH₂ CH₂ NH--),N-benzyloxycarbonylsarcosyl-N-hydroxy-5-norbornene-2,3-dicarboximideactive ester (A═ONB), R═COCH₂ N(CH₃)--), andN-benzyloxycarbonyl-L-(2-hydroxy-4-amino)butyryl-N-hydroxy-5-norbornene-2,3-dicarboximideactive ester (A═ONB, R═COCH(OH)CH₂ CH₂ NH--) where the symbol Z refersto the benzyloxycarbonyl group ##STR4## ONB refers to N-hydroxynorbornyldicarboximide and ONS refers toN-benzyloxycarbonyloxy)succinimide.

After the above illustrative couplings, the following intermediates areobtained:4-N-(N-benzyloxycarbonylglycyl)-1,2',6'-tri-N-salicyladehydeSchiff base fortimicin AM;4-N-(N-benzyloxycarbonyl-β-alanyl)-1,2',6'-tri-N-salicylaldehyde Schiffbase fortimicin AM;4-N-benzyloxycarbonylsarcosyl)-1,2',6'-tri-N-salicylaldehyde Schiff basefortimicin AM; and4-N-[N-benzyloxycarbonyl-(L-2-hydroxy-4-aminobutyryl)]-1,2',6'-triN-salicylaldehydeSchiff base fortimicin AM respectively.

It will be readily apparent to those skilled in the art that bysubstituting the appropriate R₃ or R₄ group, any of the acyl-containingintermediates for the corresponding final products can be obtained.

The Schiff base intermediates are treated with 0.2 N aqueoushydrochloric acid to cleave the Schiff base protecting groups and theresulting crude trihydrochloride salts are subjected to silica gelchromatography in a solvent system containing ammonium hydroxide whichresults in the following illustrative, partially deprotectedintermediates:4-N-(N-benzyloxycarbonylglycyl)-fortimicin AM;4-N-(N-benzyloxycarbonyl-β-alanyl)fortimicin AM;4-N-(N-benzyloxycarbonylsarcosyl)fortimicin AM; and4-N-[N-benzyloxycarbonyl-(L-2-hydroxy-4-aminobutryly)]fortimicin AM. The4-N-protected intermediates are then reacted withN-benzyloxycarbonyl-5-norbornene-2,3-dicarboximide(Z-ONB) to form thecorresponding tetra-N-protected intermediates, i.e.tetra-N-benzyloxycarbonyl-4-N-glycylfortimicin AM.

Hydrogenolysis of the tetra-N-protected intermediates over palladium oncarbon catalyst(5% Pd/C) in, for example 0.2 N hydrochloric acid inmethanol yields the desired final products, i.e. 4-N-glycylfortimicin AMtetrahydrochloride, 4-N-sarcosylfortimicin AM tetrahydrochloride, etc.

4-N-alkylation is readily accomplished by reducing the correspondingacyl, hydroxyacyl or amino-containing acyl product with diborane.

2'-N-acylation is accomplished by rearrangement of the corresponding4-N-derivative bearing the desired substituent, prepared as describedabove, by, for example, converting the stable acid addition salts to thefree bases with the use of a suitable anion exchange resin and placingthe desired 4-N-substituted free base in water which readily rearrangesthe C₄ -nitrogen substituent to the nitrogen atom attached to the C₂'-carbon. Treatment of the 2'-N-substituted fortimicin with a suitableN-acylating agent such as N-(benzyloxycarbonyloxy)succinimide, etc., asdescribed above, in a solvent system such asN,N-dimethylformamide-methanol-water, results in 6',1-di-N-protectionand the di-N-protected intermediate can then be N-acylated at the4-position as described above to provide the 4,2'- di-N-acyl derivativesof the fortimicin of this invention, using the term acyl broadly toencompass the "acyl"-containing definitions for R₃ and R₄ of Formula II.

2'-N-alkylation is achieved, as described above, by reducing theappropriate C₂ '-N-acyl substituent with a suitable reducing agent suchas diborane or a metal hydride such as lithium aluminum hydride. Theresulting 2'-N-alkyl derivatives can then be 4-N-acylated as describedabove to provide 2'-N-alkyl-4-N-acyl derivatives.

The 4,2'-di-N-alkyl derivatives can be prepared by treating theappropriate 4,2di-N-acyl derivatives, suitable N-protected, with asuitable reducing agent as described above and deblocking byhydrogenolysis.

It is to be understood that the terms acy and alkyl have, for thepurpose of the above discussion have been used as shorthand referencesto the terms "loweralkyl" and "acyl" defined on pages 3 and 4 of thespecification and to the acyl and alkyl-containing definitions for R₃and R₄ is Formula II. This shorthand reference has been used to simplifythe above discussion, not to modify the terms as defined.

The following Examples further illustrate the present invention bysetting forth the fermentation and isolation of fortimicin AM and APwhich is coproduced with fortimicin A, fortimicin B, isofortimicin,fortimicin E and a number of other minor factors.

Fortimicins AM and AP are prepared by the fermentation of Micromonosporaolivoasterospora ATCC 21819 in a suitable fermentation broth andisolated as described hereinbelow.

EXAMPLE 1 Preparation of Fermentation Broth

6000 Liters of a fermentation broth having the following composition andpH 7 before sterilization is prepared:

    ______________________________________                                        Ingredient          Weight Percent                                            ______________________________________                                        Starch              4.00                                                      Soybean meal        2.00                                                      Cornsteep liquor    0.05                                                      K.sub.2 HPO.sub.4   0.05                                                      MgSO.sub.4 . 7 H.sub.2 O                                                                          0.05                                                      KC1                 0.03                                                      CaCO.sub.3          0.1                                                       Water               to 100.00                                                 ______________________________________                                    

EXAMPLE 2 Preparation of Inoculum

Micromonospora olivoasterospora ATCC 21819 is used as a seed strain andis initially cultured in a first seed medium containing 2% glucose, 0.5%peptone, 0.5% yeast extract and 0.1% calcium carbonate (pH 7.2 beforesterilization) by inoculating one loopful of the seed strain into 10 mlof the seed medium in a 50 ml large test tube. Culturing is carried outat 30° C. for 5 days with shaking. Ten ml of the seed culture broth isthen inoculated into 30 ml of a second seed medium in a 250 mlErlenmeyer flask. The composition of the second seed medium is the sameas that of the first seed medium. The second seed culturing is carriedout at 30° C. for two days with shaking.

Then 30 ml of the second seed culture broth is inoculated into 300 ml ofa third seed medium in a two liter Erlenmeyer flask provided withbaffles. The composition of the third seed medium is the same as that ofthe first seed medium and the third seed culturing is carried out at 30°C. for 2 days with shaking. Thereafter, 1.5 liters of the third seedculture broth (corresponding to the contents of five flasks) isinoculated into 15 liters of a fourth seed medium in a 30 liter glassjar fermenter. The composition of the fourth seed medium is the same asthat of the first seed medium. Culturing in the jar fermenter is carriedout at 30° C. for two days with aeration (15 liters/min) and stirring(350 r.p.m.).

EXAMPLE 3 Production of Fortimicins AM and AP

Fifteen liters of the fourth seed culture broth of Example 2 isinoculated into 150 liters of a main fermentation medium in a 300 literstainless steel fermenter. The main fermentation medium comprises: 4%starch, 2% soybean meal, 1% corn steep liquor, 0.05% K₂ HPO₄, 0.05%MgSO₄.7H₂), 0.3% KCl and 0.1% CaCO₃ and water. (pH 7.0 beforesterilization). Culturing in the fermenter is carried out at 30° C. for4 days with aeration (80 liters/min) and stirring (150 r.p.m.).

EXAMPLE 4 Isolation of Fortimicins AM and AP

To 5000 liters of the fermentation broth, prepared as described above,is added 102 liters of a weakly acidic carboxylic (polymethacrylate)type cation exchange resin in the ammonia form, e.g. Amberlite IRC-50sold by the Rohm and Haas Company. The mixture is agitated for twohours, during which time the mixture is maintained at pH 6.6 by theaddition of sulfuric acid. The ion exchange resin is separated from thebroth by centrifugation and then added to a column and backwashed withdeionized water until free of extraneous solids. The column is washedwith water, then eluted downflow with 1 N ammonium hydroxide. Elutes ofpH 9.6 to about 11.3 are collected and concentrated under reducedpressure until excess ammonia is removed. The solution is adjusted to pH2.0 with hydrochloric acid and treated with 5% (w/v) activated carbonsuch as Pittsburgh RB carbon sold by Calgon Corporation. The solution isthen filtered through a diatomaceous earth mat and the filtrantconcentrated under reduced pressure to give a mixture of crudefortimicins and metabolites.

A portion of the crude fortimicin is chromatographed on a column ofDowex CG-50 resin eluted with 0.3 M ammonium hydroxide. Initial andfinal fractions are discarded. The median fractions are combined,adjusted to pH 2.0 with H₂ SO₄ and treated with carbon. The mixture isfiltered and the pH of the filtrate is adjusted to 6.0 with Dowex WGRresin (NH₄ ⁺ form) and the resin removed. The solution is concentratedand rechromatographed over a column of Dowex CG-50 resin eluted with0.125 N ammonium hydroxide. Initial fractions are combined and adjustedto pH 6 with sulfuric acid. Amberlite IR-124 resin (NH₄ ⁺ form) (3liters) is added and after 30 minutes filtered off and washed well withwater. The combined filtrate and washings are treated with 300 g ofPittsburg RB carbon sold by Calgon Corporation. The mixture is filtered.The precipitate is washed with water and the combined filtrate andwashings are treated with 4 liters of Dowex WGR resin (NH₄ ⁺ form) andthe resin is removed. The filtrate (at pH 6) is concentrated to aresidue.

A portion of the residue is chromatographed on a column of Bio Rex 70resin (NH₄ ⁺ form) (2.5 cm diam×40 cm) and eluted stepwise with 0.3 N,0.5 N and 1.0 N ammonium hydroxide. The eluate fractions containing thedesired components as detected by thin layer chromatography are combinedand concentrated to 2.6 g of solid. This is chromatographed on a columnof silica gel (2.5 cm diam×58 cm) packed and loaded inchloroform-methanol [1:1 (v/v)] and eluted with the lower phase of amixture of chloroform-methanol-ammonium hydroxide [1:1:1, (v/v/v)] togive a mixture containing the desired components (660 mg).

A portion (400 mg) of this is chromatographed over a column (1.1 cmdiam×60 cm) of silica gel developed with a mixture of methylenechloride-ethanol-ammonium hydroxide [1:2:1, (v/v/v)]. Initial fractionsyielded fortimicin AP (111 mg). Proton magnetic resonance spectrum indeuterium oxide with tetramethylsilane as external reference δ1.51 (3H)doublet 7'-CH₃ ; δ2.89 (3H) singlet NCH₃ δ5.61 (1H) doublet C_(1') -H.

Later fractions yielded fortimicin AM (80 mg) proton magnetic spectrummeasured similarly to that of fortimicin AP δ1.48 (3H) doublet 7'-CH₃ ;δ2.80 (3H) singlet NCH₃ and δ5.58 (1H) doublet C_(1') -H.

The following examples illustrate the present invention.

EXAMPLE 5 1,2',6'-Tri-N-salicylaldehyde Schiff base fortimicin AM

A solution of 1.3 g of fortimicin and 1.03 g of salicylaldehyde in 30 mlof methanol is refluxed and stirred for 1 hour. The solvent isevaporated under reduced pressure and the residue is dissolved in 30 mlof benzene which is likewise evaporated under reduced pressure. Thislast process is repeated six times. The residue is dried under highvacuum over KOH pellets to yield the desired product.

EXAMPLE 6 4-N-(N-Benzyloxycarbonylglycyl)-1,2',6',-tri-N salicylaldehydeSchiff base fortimicin

A solution of the above prepared 1,2'-di-N-salicylaldehyde Schiff basefortimicin (2.8 g) and N-benzyloxycarbonylglycyl-N-hydroxysuccinimideactive ester (2 g) in 25 ml of tetrahydrofuran is stirred at roomtemperature overnight. The solvent is evaporated under reduced pressureto afford a residue of about 4 g of product.

EXAMPLE 7 4-N-(N-Benzyloxycarbonylglycyl)fortimicin AM

The substance obtained in Example 6(4 g) is dissolved in 500 ml ofchloroform and the solution is shaken with 500 ml of 0.2 N aqueoushydrochloric acid. The layers are separated and the chloroform solutionis extracted with three 150 ml portions of 0.2 N aqueous hydrochloricacid. The hydrochloric acid extracts are washed in series with three 250ml portions of chloroform. The chloroform solutions are dried overanhydrous sodium sulfate, filtered, combined and evaporated to leave aresidue of nonbasic material which is not characterized.

The aqueous hydrochloric acid extracts are evaporated under reducedpressure at room temperature. The residue is dissolved in 60 ml ofmethanol and the solvent is likewise evaporated. This last process isrepeated six times. The residue is dried over potassium hydroxidepellets under high vacuum to afford crude4-N-(N-benzyloxycarbonylglycyl)fortimicin AM trihydrochloride salt.

A partial purification of the above residue(2.0 g) by chromatography on270 g of silica gel using the lower phase of a mixture ofchloroform-methanol-concentrated aqueous ammonium hydroxide[1:1:1(v/v/v)] as the eluting solvent system affords a mixturecontaining the desired product. Further chromatography of thisresidue(ca 1.4 g) on 180 g of silica gel using the lower phase of achloroform-methanol-concentrated aqueous ammonium hydroxide-watermixture[2:2:1:1(v/v/v/v)] as the solvent system leads to the separationof several components. Evaporation of the solvent from the earlychromatographic fractions leads to the isolation of nonpolar substanceswhich are not further characterized. A next group of fractions yields asmall quantity of 4,2'-di-N-(N-benzyloxycarbonylglycyl)fortimicin AMLater fractions of the chromatogram result in the desired product.

EXAMPLE 8 N-Oxybenzyloxycarbonyl-5-norbornene-2,3-dicarboximide

To an ice cooled suspension of 30 g ofN-hydroxy-5-norbornene-2,3-dicarboximide in 150 ml of water are added7.06 g of sodium hydroxide pellets over a period of 10 minutes withstirring. Methanol is added to the ice bath to bring the temperature to-5° C. and the contents of the flask are stirred for 10 minutes.Twenty-three ml of benzyloxycarbonyl chloride are then added to thestirred solution over a period of 15 minutes. The mixture is thenstirred at -5° C. for 2 hours and then at room temperature for 24 hours.The reaction mixture is extracted with 400 ml of chloroform and thechloroform extract washed with three 200 ml portions of water. Theaqueous washes are then extracted in series with four 200 ml portions ofchloroform. The chloroform extracts are dried over anhydrous magnesiumsulfate, filtered, combined and evaporated to leave a residue of 39.88g. The crude material is recrystallized from 95% ethanol. The crystalswhich form upon cooling are collected on a filter and washed withseveral small portions of cold ethanol. After drying, 29.11 g ofcrystalline product is obtained, m.p.126°-127° C. A sample isrecrystallized twice more for analysis:m.p. 126°-127° C.; IR(CDCl₃) 1800(shoulder), 1782,1732 cm⁻¹ : PMR (CDCl₃) 7.41(Z-Ar), 6.2(vinyl H), 5.31(CH₂ -Z), 3.4(H single proton), 1.7(CH₂)ppm.

Anal. Calcd. for C₁₇ H₁₅ NO₅ : C,65.17; H,4.83; N, 4.47. Found: C,65.02;H,4.82; N, 4.26.

EXAMPLE 9 Tetra-N-benzyloxycarbonyl-4-N-glycylfortimicin AM

A solution containing 1.03 g of the above prepared4-N-(N-benzyloxycarbonyl)glycylfortimicin and 2 g ofN-oxybenzyloxycarbonyl-5-norbornene-2,3-dicarbonximide in 56 ml ofmethanol are stirred at room temperature overnight. Evaporation of thesolvent under reduced pressure leaves a residue of about 3 g of crudereactoin mixture. The latter is purified by repeated silica gel columnchromatography using benzene-methanol-ethanol [1170:34:136(v/v/v)] andbenzene-chloroform-ethyl acetate-n-propanol[13:16:8:3(v/v/v/v)] as theeluting systems.

Combination of the appropriate fractions following the minorunidentified somponenet in the original chromatograms and evaporation ofthe solvents leave a residue of the desired product. An analyticalsample can be obtained by rechromatography of a part of the aboveproduct on silica gel using benzene-methanol-ethanol[1170:36:136(v/v/v)]as the eluant.

EXAMPLE 10 4-N-GlycylfortimicinAM tetrahydrochloride salt

A solution of 0.5 g of tetra-N-benzyloxycarbonyl-4-N-lycylortimicinAM in34 ml of 0.2 N hydrochloric acid in methanol and 16 ml of methanol ishydrogenolyzed over 5% Pd/C for 4 hours. The catalyst is collected on afilter ans washed with methanol. The filtrate is evaporated underreduced pressure, the residue redissolved in methanol and this solventevaporated. This last procedure is repeated six times to yield thedesired product after drying in vacuo over potassium hydroxide pellets.

EXAMPLE 11 4-N-(N-Benzyloxycarbonyl-β-alanyl)fortimicin AP

A solution of 2.579 g of compound of Example 5 and 2.5 g of theN-benzyloxycarbonyl-β- alanyl ester ofN-hydroxy-5-norbornene-2,3-dicarboximide, prepared by the method of M.Jujino et al., Chem Pharm. Bull. Japan, 22, 1857(1974), in 30 ml oftetrahydrofuran is stirred at room temperature for 25 hours. Evaporationof the solvent under reduced pressure yields about 5 g of crude product.

This material is dissolved in 500 ml of chloroform and the solution isshaken with 500 ml of 0.2 N hydrochloric acid. The layers are separatedand the chloroform solution is extracted with three 150 ml portions of0.2 N hydrochloric acid. The acid extracts are washed in series withthree 25 ml portions of chloroform. The chloroform washed are dried overanhydrous magnesium sulfate, filtered, combined and evaporated to yielda non-basic material. The acidic extracts are combined and evaporatedunder reduced pressure at low temperature. The residue is dissolved in30 ml of methanol which is also evaporated. This last process isrepeated six times to yield a residue of reaction products as thehydrochloride salts after drying in vacuo over potassium hydroxide. Theabove hydrochloride salts are chromatographed on 170 g of silica gelusing the lower layer of a chloroform-methanol-concentrated ammoniumhydroxide mixture[1:1:1(v/v/v)] as the eluting solvent. Combination ofthe fractions containing the desired product and evaporation of thesolvents affords a residue which is rechromatographed on 170 g of silicagel using the lower phase of a chloroform-methanol-concentrated ammoniumhydroxide-water [2:2:1:1(v/v/v/v)] mixture as the eluting solvent. Aftercombination and evaporation of the solvents from the appropriatefractions, the desired product is obtained

EXAMPLE 12 Tetra-N-benzyloxycarbonyl-4-N-β-alanylfortimicin AP

A solution of 0.75 g of the above prepared4-N-(N-benzyloxycarbonyl-β-alanylfortimicin AP and 1.440 g ofN-oxybenzyloxycarbonyl-5-norbornene-2,3-dicarboximide in 40 ml ofmethanol is stirred at room temperature for 24 hours. Evaporation of thesolvent leaves a residue which is chromatographed on 180 ml of silicagel using a methylene chloride-methanol-concentrated ammonium hydroxidemixture [185:15:2(v/v/v)] as the eluent. Combination of the fractionscontaining the deisred usbstance and evaporation of the solvent leaves apartially purified product. Repeated chromatography of this sample onsilica gel using benzene-methanol-ethanol[1170:34:136(v/v/v)], methylenechloride-methanol-concentrated ammonium hydroxide[185:15:2(v/v/v)]yields the desired product.

EXAMPLE 13 4-N-β-Alanylfortimicin AP tetrahydrochloride

A solution of 0.35 g of the compound of Example 12 in 30 ml of 0.2 Nhydrochloric acid in methanol and 20 ml of methanol is hydrogenolyzedover 0.36 g of 5% Pd/C for 4 hours. The catalyst is collected on afilter and washed with several small portions of methanol. The filtrateis evaporated under reduced pressure and the residue is dissolved in 30ml of methanol which is likewise evaporated under reduced pressure. Thislast process is repeated six times to afford a residue of about 0.2 g ofthe desired product after drying in vacuo over potassium hydroxidepellets.

EXAMPLE 14 N-Benzyloxycarbonylsarcosyl active ester ofN-hydroxy-5-norbornene-2,3-dicarboximide

To a solution of 4.471 g of N-benzyloxycarbonylsarcosine and 3.754 g ofN-hydroxy-5-norbornene-2,3-dicarboximide in 15 ml of tetrahydrofuran and15 ml of dioxane is added N,N-dicyclohexylcarbodiimide in 2 ml oftetrahydrofuran and 2 ml of dioxane according to the method of M. Fujinoet al., Chem Pharm. Bull.,Japan, 22, 1857(1974). The mixture is stirredat room temperature overnight. The dicyclohexylurea which precipitatesfrom the reaction mixture is collected on a filter and washed with atotal of 20 ml of tetrahydrofuran-dioxane[1:1(v/v)]. Evaporation of thesolvent from the filtrate affords 8.523 g of crude product. Thesubstance is recrystallized from isopropanol to yield 4.294 g of theactive ester, m.p. 75°-80° C. Concentration of the mother liquors yieldsan additional 4.125 g of less pure product, m.p. 69°-73° C.

A portion of the first crop is recrystallized for analysis: m.p. 80°-82°C.; IR(CDCl₃) 1821,1774,1725,1700(shoulder) cm⁻¹ ; NMR (CDCl₃)7.32(Ar-Z), 6.17(vinyl), 5.13(CH₂ -Z), 4.3(sar-CH₂), 3.35(single H),3.0(sar-CH₃), 1.64(-CH₂) ppm.

Anal. Calcd. for C₂₀ H₂₀ N₂ O₆ : C, 62.49; H,5.24; N, 7.29. Found: C,62.65; H, 5.28;N, 7.24.

EXAMPLE 15 4-N-(N-Benzyloxycarbonylsarcosyl)fortimicin AM

A solution of 2.4 g of the compound of Example 5 and 2.5 g of theN-benzyloxycarbonylsarcosyl active ester ofN-hydroxy-5-norbornene-2,3-dicarboximide of Example 14 in 25 ml oftetrahydrofuran is stirred for 24 hours at room temperature. Evaporationof the solvent under reduced pressure affords a residue of crudeproduct. The residue is taken up in 500 ml of chloroform and thesolution is shaken with 500 ml of 0.2 N aqueous hydrochloric acid. Thelayers are separated and the chloroform solution is extracted with three150 ml portions of 0.2 N hydrochloric acid. The acid extracts are washedin series with three 250 ml portions of chloroform. The chloroformwashes are dried over anhydrous sodium sulfate, filtered, combined andevaporated to leave a non-basic residue.

The acidic extracts are combined and evaporated under reduced pressureat low temperature. The residue is taken up in 30 ml of methanol whichis likewise evaporated and the procedure repeated six times. Theresulting residue is dried in vacuo over potassium hydroxide pellets.

The above residue is chromatographed on 160 g of silica gel using thelower phase of a chloroform-methanol-concentrated ammonium hydroxidemixture[1:1:1(v/v/v)] as the eluting solvent. The fractions containingthe desired product are combined and the solvents evaporated leavingcrude product which is rechromatographed on 160 g of silica gel usingthe lower phase of a chloroform-methanol-concentrated ammoniumhydroxide-water mixture[2:2:1:1(v/v/v/v)] as the eluting solvent. Aftercombination of the appropriate fractions and evaporation of the solventsthe desired product is obtained.

EXAMPLE 16 Tetra-N-benzyloxycarbonyl-4-N-sarcosylfortimicin AM

A solution of 0.15 g of the product of Example 15 and 0.3 g ofN-oxybenzyloxycarbonyl-5-norbornene 2,3-dicarboximide in 10 ml ofmethanol is stirred at room temperature overnight. Evaporation of thesolvent under reduced pressure affords a residue which ischromatographed on 70 g of silica gel using a benzene-methanol-ethanolmixture[1170:34:136(v/v/v)] as the eluent. Combination of theappropriate fractions and evaporation of the solvent yields partiallypurified tetra N-benzyloxycarbonyl-4-n-sarcosylfortimicin AM. A secondchromatogram of this substance on 50 g of silica gel, employing the samesolvent system yields the desired product in pure form.

EXAMPLE 17 4-N-Sarcosylfortimicin AM tetrahydrochloride

A solution of 0.15 g of the compound of Example 16 in 12 ml of 0.2 Nhydrochloric acid in methanol and 23 ml of methanol is hydrogenolyzedover 0.150 g of a 5% Pd/C catalyst for 4 hours. The catalyst iscollected on a filter and washed with methanol. The filtrate isevaporated to dryness under reduced pressure and the residue isdissolved in 20 ml of methanol which is likewise evaporated. The lastprocedure is repeated six times. The residue is dried in vacuo overpotassium hydroxide pellets to afford the desired product.

EXAMPLE 184-N-[N-Benzyloxycarbonyl-(L-2-hydroxy-4-aminobutyryl)]-fortimicin AM

The N-hydroxy-5-norbornene-2,3-dicarboximide active ester ofL-N-benzyloxycarbonyl-2-hydroxy-4-aminobutyric acid is preparedaccording to the method of M. Fujino et al. supra. To an ice coldsolution of 1.645 g of L-N-benzyloxycarbonyl-2-hydroxy-4-aminobutyricacid and 1.182 g of N-hydroxy-5-norbornene-2,3-dicarboximide in 16 ml oftetrahydrofuran-dioxane [1:1(v/v)], are added, with stirring, 1.374 g ofN,N-dicyclohexylcarbodiimide and 5 ml oftetrahydrofurandioxane[1:1(v/v)]. The mixture is stirred at 0° C. for 50minutes and then at room temperature for 3 hours.

The N,N-dicyclohexylurea produced by the above reaction is collected ona filter and washed with 10 ml of tetrahydrofuran-dioxane[1:1(v/v)]. Thefiltrate is collected in a flask containing 2.0 g of the compound ofExample 5. The reaction mixture is stirred at room temperature for 20hours. Evaporation of the solvent leaves crude product.

Five and one-half grams of the crude product is dissolved in 500 ml ofchloroform and the solution is shaken with 500 ml of 0.2 N hydrochloricacid. The chloroform phase is separated and extracted with three 150 mlportions of 0.2 N hydrochloric acid. The aqueous phase are washed inseries with three 250 ml portions of chloroform. The chloroformsolutions are dried over anhydrous sodium sulfate, filtered, combined,and evaporated to afford a residue of non-basic substances.

The aqueous extracts are combined and evaporated in vacuo at lowtemperature. The residue is dissolved in 30 ml of methanol and thesolvent evaporated. This last process is repeated six times and theresulting material is dried over potassium hydroxide pellets in vacuo toyield a residue of crude product.

3.5 g of the crude product are chromatographed on 270 g of silica gelusing the lower phase of a chloroform-methanol-concentrated ammoniumhydroxide[1:1:1(v/v/v)] mixture as the eluent. The fractions containingthe desired substance are combined and evaporation of the solvent yieldsa residue of partially purified product which is rechromatographed on180 g of silica gel using the lower phase of achloroform-methanol-concentrated ammonium hydroxide-water[2:2:1:1(v/v/v/v)] mixture as the eluent. Combination and evaporation ofthe active fractions affords the desired product.

EXAMPLE 19 Tetra-Nbenzyloxycarbonyl-4-N-(L-2-hydroxy-4-aminobutyryl)-fortimicin AM

A solution of 0.8 g of the above prepared substance and 1.5 g ofN-oxybenzyloxycarbonyl-5-norbornene-2,3-dicarboximide in 40 ml ofmethanol is stirred at room temperature overnight. Evaporation of thesolvent from the reaction mixture under reduced pressure affords aresidue of about 2 g of crude product which is chromatographed on 180 gof silica gel using a mixture of methylenechloride-methanol-concentrated ammonium hydroxide[185:15:2(v/v/v)] asthe eluent. The early fractions of the chromatogram contained thedesired substance contaminated with less polar components. From thesubsequent fractions, the desired product is isolated after evaporationof the solvent. Repeated silica gel chromatography of the residue of theearly fractions above using benzene-methanol-ethanol-acetic acid[1170:35:135:10(v/v/v/v)] and ethyl acetate-ethanol[98:2(v/v)] mixturesaffords additional product. After two more chromatograms on silica gelusing ethyl acetate-ethanol[98:2(v/v)] as the eluent, the desiredproduct is obtained in pure form.

EXAMPLE 20 4-N-(L-2═Hydroxy-4-aminobutyryl)fortimicin AMtetrahydrochloride

A solution of about 0.2 g of the above prepared compound in 16.3 ml of0.2 N hydrochloric acid and 8.7 ml of methanol is hydrogenolyzed over a0.2 g of 5% Pd/C for 4 hours. The catalyst is collected on a filter andwashed with methanol. The filtrate is evaporated under reduced pressureand the residue is dissolved in 10 ml of methanol which is likewiseevaporated. This last process was repeated six times and the resultingmaterial is dried under high vacuum over potassium hydroxide pellets toafford the desired material.

EXAMPLE 21 Tetra-N-benzyloxycarbonyl-4,2'-N,N'-diglycylfortimicin AM

A solution of the substance contained in the chromatographic fractionspreceeding the final product of Example 7 and 0.128 g ofN-oxybenzyloxycarbonyl-5-norbornene -2,3-dicarboximide in 3 ml ofmethanol is stirred at room temperature overnight. Evaporation of thesolvent affords a residue which is chromatographed in silica gel usingbenzene-ethanol-isopropanol[9:1:1(v/v/v)] as the eluting solvent.Combination of the appropriate fraction and evaporation of the solventleaves a residue which is rechromatographed on silica gel using ethylacetate-methanol[98:2 v/v)] as the eluting solvent to afford the desiredproduct.

EXAMPLE 22 4,2'-N,N'-Diglycylfortimicin AM tetrahydrochloride

A solution of 0.75 g of the above prepared compound in 6 ml of 0.02 Nhydrochloric acid in methanol is hydrogenolyzed over 0.08 g of 5% Pd/Cfor four hours. The catalyst is collected on a filter and washed withseveral portions of methanol. The filtrate is evaporated under reducedpressure and the residue redissolved in 15 ml of methanol which islikewise evaporated. This last procedure is repeated six times and thedesired product dried in vacuo pver potassium hydroxide pellets.

EXAMPLE 23 4N-(β-Aminoethyl)fortimicin AM

A stirring solution of 4-N-glycylfortimicin (2.0 g) in tetrahydrofuran(80 ml) is treated with 1.22 g of lithium aluminum hydride. The stirringreaction mixture is refluxed for 20 hours and then the excess lithiumaluminum hydride is consumed by the careful addition of water. Theinsoluble material is sedimented by centrifugation. The pellet issuspended in 50 ml of water and centrifuged. The combined supernatantsare taken to dryness under reduced pressure to yield crude product whichis chromatographed on a column (2.0×40 cm) of cation exchange resin,carboxylic type, e.g., Bio-Rad Laboratories, Bio-Rex 70,100-200 mesh,ammonia form, and eluted with a gradient of water to 1 N ammoniumhydroxide. Fractions containing the desired product are concentrated toa small volume and lyophilized to give the desired product.

The in vitro antibiotic activity is determined by a two-fold agardilution method using 10 ml per petri plate of Mueller-Hinton agar. Theagar is inoculated with one loopful (0.001 ml loop) of a 1:10 dilutionof a 24 hour broth culture of the indicated test organism and incubatedat 37° C. for 24 hours.

The compounds of this invention are active as systemic antibiotics wheninjected by parenteral routes of administration, i.e., by theintramuscular, intravenous, intraperitoneal or subcutaneous routes ofadministration. The compounds can also be administered orally in thoseinstances where it is desirable to sterilize the intestinal tract andcan also be applied topically or in suppository form.

Preparations according to this invention for parenteral administrationinclude sterile aqueous or non-aqueous solutions, suspensions, oremulsions. Examples of non-aqueous solvents or vehicles are propyleneglycol, polyethylene glycol, vegetable oils such as olive oil, andinjectable organic esters such as ethyl oleate. Such dosage forms mayalso contain adjuvants such as preserving, wetting, emulsifying anddispersing agents. They may be sterilized, by, for example, filtrationthrough a bacteria-retaining filter, by incorporating sterilizing agentsinto the compositions and the like. They can also be manufactured in theform of sterile solid compositions which can be dissolved in sterilewater, or some other sterile injectable medium immediately before use.

Solid dosage forms for oral administration including capsules tablets,pills, powders and granules. In such solid dosage forms, the activecompound is admixed with at least one inert diluent such as sucrose,lactose or starch. Such dosage forms can also comprise, as is normalpractice, additional substances other than inert diluents, e.g.,lubricating agents such as magnesium stearate. In the case of capsules,tablets and pills, the dosage forms may also comprise buffering agents.Tablets and pills can additionally be prepared with enteric coatings.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups and elixirscontaining inert diluents commonly used in the art, such as water.Besides, such inert diluents compositions can also include adjuvants,such as wetting agents, emulsifying and suspending agents, andsweetening, flavoring and perfuming agents.

The dosage of active ingredient in the compositions of this inventionmay be varied; however, it is necessary that the amount of the activeingredient shall be such that a suitable dosage form is obtained. Theselected dosage depends upon the desired therapeutic effect, on theroute of administration, and on the duration of the treatment.Generally, dosage levels of between 20 to 40 mg/kg of body weight daily,based on lean body weight are administered to a mammalian patientsuffering from an infection caused by susceptible organism.

The invention claimed is:
 1. A fortimicin selected from the groupconsisting of 4-N, 2'-N and 4,2'-di-N-derivatives of fortimicin AM andfortimicin AP represented by the formula: ##STR5## wherein: R₁ ishydrogen or hydroxy; R₂ is hydroxy when R₁ is hydrogen and is hydrogenwhen R₁ is hydroxy; and R₃ and R₄ are the same or different members ofthe group consisting of acyl, aminoacyl, diaminoacyl,N-loweralkylaminoacyl, N,N-diloweralkylaminoacyl, hydroxy-substitutedaminoacyl, loweralkyl, aminoloweralkyl, diaminoloweralkyl,hydroxyloweralkyl, N-loweralkylaminoloweralkyl,N,N-diloweralkylaminoloweralkyl, N-loweralkylaminohydroxyloweralkyl,N,N-diloweralkylaminohydroxyloweralkyl, with the limitation that R₃cannot be hydrogen when R₄ is hydrogen; and the pharmaceuticallyacceptable salts thereof.
 2. A compound of claim 1 wherein R₁ ishydrogen and R₂ is hydroxy.
 3. A compound of claim 2 wherein R₄ ishydrogen.
 4. A compound of claim 3: 4-N-glycylfortimicin AM or apharmaceutically acceptable salt thereof.
 5. A compound of claim 3:4-N-sarcosylfortimicin AM or a pharmaceutically acceptable salt thereof.6. A compound of claim 3: 4-N-beta-alanylfortimicin AM or apharmaceutically acceptable salt thereof.
 7. A compound of claim 3:4-N-(L-2-hydroxy-4-aminobutyryl)fortimicin AM or a pharmaceuticallyacceptable salt thereof.
 8. A compound of claim 3:4-N-beta-aminoethylfortimicin AM or a pharmaceutically acceptable saltthereof.
 9. A compound of claim 2 wherein R₃ is hydrogen.
 10. A compoundof claim 9: 2'-N-glycylfortimicin AM or a pharmaceutically acceptablesalt thereof.
 11. A compound of claim 2 wherein neither R₃ or R₄ arehydrogen.
 12. A compound of claim 11: 4,2'-di-N-glycylfortimicin AM or apharmaceutically acceptable salt thereof.
 13. A compound of claim 1wherein R₁ is hydroxy and R₂ is hydrogen.
 14. A compound of claim 13wherein R₄ is hydrogen.
 15. A compound of claim 14: 4-N-glycylfortimicinAP or a pharmaceutically acceptable salt thereof.
 16. A compound ofclaim 14: 4-N-sarcosylfortimicin AP or a pharmaceutically acceptablesalt thereof.
 17. A compound of claim 14: 4-N-beta-alanylfortimicin APor a pharmaceutically acceptable salt thereof.
 18. A compound of claim14: 4-N-(L-2-hydroxy-4-aminobutyryl)fortimicin AP or a pharmaceuticallyacceptable salt thereof.
 19. A compound of claim 14:4-N-beta-aminoethylfortimicin AP or a pharmaceutically acceptable saltthereof.
 20. A compound of claim 13 wherein R₃ is hydrogen.
 21. Acompound of claim 20: 2'-N-glycylfortimicin AP or a pharmaceuticallyacceptable salt thereof.
 22. A compound of claim 13 wherein neither R₃nor R₄ are hydrogen.
 23. A compound of claim 22:4.2'-di-N-glycylfortimicin AP or a pharmaceutically acceptable saltthereof.
 24. A pharmaceutical composition comprising an antibacteriallyeffective amount of a compound of claim 1 and a pharmaceuticallyacceptable carrier or diluent.